Advanced Pharmaceutical Bulletin, 2013, 3(2), 409-413

doi: http://dx.doi.org/10.5681/apb.2013.065

http://apb.tbzmed.ac.ir/

*Corresponding author: Ruchi Jain, Research Scholar, Suresh Gyan Vihar University, Jagatpura Mahal, Jaipur, Rajasthan, India-302025.

Email: jainruchi02@gmail.com

Copyright © 2013 by Tabriz University of Medical Sciences

A Novel Approach using Hydrotropic Solubalization Technique for

Quantitative Estimation of Entacapone in Bulk Drug and Dosage Form

Ruchi Jain1*, Nilesh Jain

2, Deepak Kumar Jain

2, Surendra Kumar Jain

2

1 Suresh Gyan Vihar University, Jaipur, Rajasthan, India-302025.

2 Sagar Institute of Research & Technology-Pharmacy, Ayodhya Bypass Road Bhopal, Madhya Pradesh, India – 462041.

Introduction

Entacapone (ENT) is chemically (E)-2-cyano-3-(3, 4-

dihydroxy-5-nitrophenyl)-N, N-diethyl-2-propenamide

(Figure 1), is a drug that functions as a catechol-O-

methyl transferase (COMT) inhibitor, used in the

treatment of Parkinson’s disease. It is a member of the

class of nitrocatechols.1,2 The drug is not official in any

pharmacopoeia. Literature survey revealed few HPLC

methods3,4 has been reported for the determination of

ENT in biological fluids. The reported methods for the

determination of ENT in tablets includes HPLC5-11 and

spectrophotometric methods.12,13

As the environmental pollution it is necessary to

preclude the use of organic solvents for analysis of

drug. Various techniques have been employed to

enhance the aqueous solubility and hydrotropy is one

of them. Hydrotropic solubilization is the phenomenon

by which aqueous solubility of poorly water soluble

drugs and insoluble drugs increases. Maheshwari and

Jain et al has used sodium salicylate, sodium benzoate,

urea, nicotinamide, sodium citrate and sodium acetate

as the most common examples of hydrotropic agents

utilized to increase the water solubility of drug.14-19

Various organic solvents such as methanol, chloroform,

dimethyl formamide and acetonitrile have been

employed for solubilization of poorly water-soluble

drugs to carry out spectrophotometric analysis.

Drawbacks of organic solvents include their higher

cost, toxicity and pollution. Hydrotropic solution may

be a proper choice to preclude the use of organic

solvents. Therefore, it was thought worthwhile to

employ this hydrotropic solution to extract out the drug

from fine powder of tablets to carry out

spectrophotometric estimation. Present work

emphasizes on the quantitative estimation of ENT in

their dosage form by UV Spectroscopic methods.

Materials and Methods

Instrument

UV-Visible double beam spectrophotometer, Shimadzu

model-1700 having spectral bandwidth 3 nm and of

wavelength accuracy ±1 nm, with 1cm quartz cells was

used.

Reagents and chemicals

Analytical pure sample of ENT was supplied as gift

sample from Sun Pharmaceuticals Ind. Ltd. Urea

obtained from Merck Chemical Division, Mumbai.

A R T I C L E I N F O A B S T R A C T

Article Type:

Research Article

Article History:

Received: 14 March 2013

Revised: 6 May 2013

Accepted: 8 May 2013

ePublished: 20 August 2013

Keywords:

Entacapone

Urea

Ecofriendly

Hydrotropic solubilizing agents

Purpose: Analysis of drug utilized the organic solvent which are costlier, toxic and

causing environment pollution. Hydrotropic solution may be a proper choice to preclude

the use of organic solvents so that a simple, accurate, novel, safe and precise method has

been developed for estimation of poorly water soluble drug Entacapone (Water

Solubility-7.97e-02 g/l). Methods: Solubility of entacapone is increased by using 8M

Urea as hydrotropic agent. There was more than 67 fold solubility enhanced in

hydrotropic solution as compare with distilled water. The entacapone (ENT) shows the

maximum absorbance at 378 nm. At this wavelength hydrotropic agent and other tablet

excipients do not shows any significant interference in the spectrophotometric assay.

Results: The developed method was found to be linear in the range of 4-20 μg/ml with

correlation coefficient (r2) of 0.9998. The mean percent label claims of tablets of ENT in

tablet dosage form estimated by the proposed method were found to be 99.17±0.63. The

developed methods were validated according to ICH guidelines and values of accuracy,

precision and other statistical analysis were found to be in good accordance with the

prescribed values. Conclusion: As hydrotropic agent used in the proposed method so

this method is Ecofriendly and it can be used in routine quantitative analysis of drug in

bulk drug and dosage form in industries.

410 | Advanced Pharmaceutical Bulletin, 2013, 3(2), 409-413 Copyright © 2013 by Tabriz University of Medical Sciences

Jain et al.

Reverse Osmosis (R.O.) Water was used throughout the

study.

Figure 1. Chemical structure of ENT

Preliminary solubility studies of drugs

An excess amount of drug was added to a screw capped

25 ml of volumetric flask containing different aqueous

systems viz. distilled water, different combination of

hydrotropic agent. The volumetric flasks were shaken

mechanically for 12 hrs at 25±1°C in a mechanical

shaker. These solutions were allowed to equilibrate for

next 24 hrs and then centrifuged for 5 min at 2000 rpm.

The supernatant liquid was taken for appropriate

dilution after filtered through whatman filter paper

no.41 and analyzed spectrophotometrically against

corresponding solvent blank. After analysis, it was

found that the enhancement in the solubility of ENT

was to be more than and 67 folds in 8 M Urea as

compared to solubility studies in other solvents.

Selection of hydrotropic agent

ENT was scanned in hydrotropic agent in the spectrum

mode over the UV range (200-400) and 8 M Urea as

hydrotropic agent were found to be most appropriate

because:

ENT is soluble in it (67 fold enhancement of

solubility)

ENT is stable in hydrotropic agent (as shown in

Figure 2)

ENT exhibit good spectral characteristics in it.

Urea solution has no interference with the λmax

of ENT i.e 378nm.

Figure 2. Spectra of ENT in 8 M Urea as Hydrotropic Agent

Establishment of stability profile

Stability of ENT was observed by dissolving in 8 M

Urea as hydrotropic agent. Solution of ENT was

prepared in the conc. of 12 g/ml and scanned under

time scan for 30 min. Spectra of drug under time scan

shows that drug are stable in hydrotropic solution.

Linearity range and calibration graph

Preparation of Standard Stock Solution (Stock-A)

Accurately weighed 100 mg of the ENT was

transferred in to 100 ml volumetric flask containing 80

ml of hydrotropic agent and the flask was sonicated for

about 10 min to solubilize the drug and the volume was

made up to the mark with mixed hydrotropic agent to

get a concentration of 1000 µg/ml (Stock-A).

Preparation of Working Standard Solution

The standard solution (1000 µg/ml) was further diluted

with distilled water to obtain 4, 8, 12, 16 and 20µg /ml

solution and absorbance were noted at 378 nm against

distilled water as blank.

Analysis of Marketed Formulation

Marketed formulation Entacom (Intas Pharmaceuticals)

was selected for tablet analysis, i. e containing 200 mg

ENT. Twenty tablets were accurately weighed, average

weight determined and ground to fine powder. An

accurately weighed quantity of powder equivalent to

100 mg of ENT was transferred into 100 ml volumetric

flask containing 80 ml of hydrotropic solution. The

flask was sonicated for about 20 min to solublize the

drug; volume was adjusted to mark with hydrotropic

agent and filtered through whatman filter paper no. 41.

The Absorbance of sample solutions was analyzed on

UV spectrophotometer at 378 nm against R.O. water as

blank.

Validation Parameters

The developed method was validated as per ICH

guidelines (Linearity, Accuracy, Precision and

Robustness).20

Linearity

Linearity of ENT was established by response ratios of

drug. Response ratio of drug was calculated by dividing

the absorbance with respective concentration.

Accuracy

To check the degree of accuracy of the method,

recovery studies were performed in triplicate by

standard addition method at 80%, 100% and 120%. In

preanalyzed tablet solution, a definite amount of drug

was added and then its recovery was studied. These

studies were performed in by adding fixed amount of

pure drug solution to the final dilution while varying

the concentration of tablet sample solution in the final

dilution.

| 411 Advanced Pharmaceutical Bulletin, 2013, 3(2), 409-413 Copyright © 2013 by Tabriz University of Medical Sciences

Hydrotropic Solubalization Technique for Estimation of Entacapone

Precision

Precision of the methods was studied at three level as at

repeatability, intermediate precision (Day to Day and

analyst to analyst) and reproducibility.

Repeatability was performed by analyzing same 5

concentrations of drug for 5 times. Day to Day was

performed by analyzing 5 different concentration of the

drug for three days in a week.

Reproducibility was performed by analyzing same

concentration of drugs for five times in different lab.

Results and Discussions

Based on the solubility, stability and spectral

characteristics of the drug, 8M Urea was selected as

hydrotropic agent. There was more than 67 fold

solubility enhanced in hydrotropic solution as compare

with distilled water. After solubilizing the Entacapone

in selected hydrotropic agent, it was scanned in

spectrum mode and the working wavelength for the

estimation, considering the reproducibility and

variability was found to be 378 nm. Spectra of ENT is

shown in Figure 2, Calibration curve was plotted

between concentrations versus absorbance Figure 3.

Observation of linearity data has reported in the Table

1. The Result of their optical characteristics has been

reported in Table 2.The developed method was found

to be linear in the range of 4-20 μg/ml with linear

equation was Y=0.098X + 0.011 and correlation

coefficient (r2) of 0.9998. Drug content of tablet

formulation was calculated using calibration curve and

values are reported in Table 3. The mean percent label

claims of tablets of ENT in formulation-I estimated by

the proposed method were found to be 99.17±0.63.

These values are close to 100, indicating the accuracy

of the proposed analytical method.

Figure 3. Calibration Curve of ENT at 378 nm in 8 M Urea

Table 1. Linearity ENT at λmax =378 nm in 8 M Urea

Standard Conc. (g/ml) Rep-1 Rep-2 Rep-3 Rep-4 Rep-5 Mean

0 0 0 0 0 0 0

4 0.403 0.408 0.41 0.411 0.401 0.4066

8 0.821 0.823 0.812 0.841 0.842 0.8278

12 1.182 1.193 1.195 1.184 1.185 1.1878

16 1.586 1.593 1.554 1.579 1.599 1.5822

20 1.976 1.996 1.99 1.986 1.988 1.9872

Correlation Coefficient (r2) - - - - - 0.9998

Slope (m) - - - - - 0.0987

Intercept (c) - - - - - 0.0113

Table 2. Optical Characteristic and Linearity Data of ENT in 8

M Urea

S. No. Parameter 8 M Urea as

Hydrotropic Agent

1 Working λ 378 nm

2 Beer’s law limit (μg/ml) 4-20

3 Correlation Coefficient (r2)* 0.9998

4 Slope (m)* 0.098

5 Intercept (c)* 0.011

6 Number of samples (n) 25

*Average of 5 determination of 5 concentrations

Result of Validation Parameters

Linearity

Linearity was established in the range of 4-20μg/ml and

it was reported as response ratio; Table 4. Then a graph

was plotted between concentration and response ratio

(Figure 4) which assure the linearity of the method.

Accuracy

The values of mean percentage recoveries were also

found to show variability in ranging from 97.83±1.03

to 98.92±0.82%. Low values of standard deviation,

percent coefficient of variation and standard error

further validated the proposed method (Table 5). All

these values were very close to 100.

412 | Advanced Pharmaceutical Bulletin, 2013, 3(2), 409-413 Copyright © 2013 by Tabriz University of Medical Sciences

Jain et al.

Table 3. Results and Statistical Parameters for Entacom 200 mg Tablet Analysis using 8M Urea

Drug Label Claim (mg) Amount Found (mg) % MEAN* S.D.* %COV* Std. Error*

Entacom200 200 198.67 99.33 0.26 0.262 0.048

Entacom200 200 198.43 99.21 0.73 0.736 0.133

Entacom200 200 197.93 98.96 0.89 0.899 0.163

Mean - 198.34 99.17 0.63 0.632 0.115

*Average of five in 3 replicates determination

Table 4. Response Ratio of ENT in Hydrotropic Solution

S. No. 8 M Urea as Hydrotropic Agent

Conc. (µg/ml) ABS Response Ratio

1. 4 0.402 0.10

2. 8 0.81 0.10

3. 12 1.194 0.10

4. 16 1.584 0.10

5. 20 1.99 0.10

Figure 4. Response Ratio Curve of ENT in 8 M Urea

Table 5. Result of Recovery Studies of Tablet Formulation with Statically Evaluation

Drug QC Conc. (μg/ml) Recovery Level %

(Amount Drug Added) Amount of Drug Found

(Mean±SD)* % RSD

ENT 10 80 98.47±1.23 0.332

- - 100 98.39±1.08 0.447

- - 120 97.83±1.03 0.40

ENT 12 80 98.86±0.63 0.90

- - 100 98.92±0.82 0.30

- - 120 98.29±0.74 0.63

*Average of five determination

Precision

Result of precision at different level were found be

within acceptable limits (RSD<2). The results have

been reported in Table 6. Presence of hydrotropic agent

do not shows any significant interference in the

spectrophotometric assay thus further confirming the

applicability and reproducibility of the developed

method.

Table 6. Result of Precision of ENT

- Validation Parameter Percentage Mean ± S.D*. (n=6) Percentage RSD

With 8 M Urea as Hydrotropic Agent Repeatability 98.64±1.31 1.33

Intermediate Precision - -

- Day to Day 98.92±1.42 1.435

- Analyst to Analyst 98.74±0.86 0.870

- Reproducibility 98.39±0.70 0.711

- - - -

* Mean of fifteen determinations (3 replicates at 5 concentrations level)

| 413 Advanced Pharmaceutical Bulletin, 2013, 3(2), 409-413 Copyright © 2013 by Tabriz University of Medical Sciences

Hydrotropic Solubalization Technique for Estimation of Entacapone

Conclusion

Hence, it is concluded that the proposed methods are

new, simple, cost effective, accurate, safe and precise

and can be successfully employed in the routine

analysis of Entacapone in bulk drug sample and tablet

dosage form. Advantage of these methods is that the

organic solvent is not essential for the analysis and

there was no interference of 8 M urea during the

estimation. There is a good scope for other poorly

water‐soluble drugs which may be tried to get

solubilized in 2 M urea solution (as hydrotropic agent)

to carry out their spectrophotometric analysis excluding

the use of costlier and unsafe organic solvents.

Conflict of Interest The authors report no conflicts of interest.

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